Stroke remains the leading cause of long-term disability in the US. To enhance post stroke functional recovery, appropriate rehabilitation strategy is in dire need. The concept of neurovascular unit and the multiple mechanisms involved in secondary injury is changing our approach in stroke therapy. Following focal cerebral ischemia, although the tissue perfusion in the peri-infarct cortex improved gradually in a distance-dependent manner, it is never fully restored even months after. It is evident that the recovery of local hemodynamics also affected the recovery of spine density, and ultimately, synaptic plasticity. Our preliminary results establish that functional restoration induced by AAV-Netrin-1 gene therapy is associated with an increase in vascular density in the peri-infarct cortex, which could be related to netrin-1's proangiogenic effect or prevention of secondary injury. Based on these promising results and the diverse effects of netrin-1 in anti-inflammation and neuroplasticity induction, we would like to extend our study to further investigate the therapeutic efficacy of netrin-1 and underlying signaling mechanisms in reducing inflammation and promoting neuroplasticity in the context of brain ischemia and functional recovery. The following specific aims are proposed: Aim 1. Test the hypothesis that netrin-1 gene transfer reduces post stroke chronic neuroinflammation via adenosine 2B receptor. To distinguish the contribution of blood versus endothelial A2B receptors, we will use bone marrow transplants to create chimeric mice. Aim 2 will test the hypothesis that netrin-1 gene transfer enhances neural stem cell migration in the ischemic peri-infarct and white matter via specific netrin-1 receptors. Aim 3 will test the hypothesis that netrin-1 gene transfer augments the neuroplasticity inducing effect of amphetamine and rehabilitation. Proof-of-concept data collected here may lead to the development of netrin-1 as a novel target for stroke therapy in promoting functional recovery. PUBLIC HEALTH RELEVANCE: Stroke remains the leading cause of long-term disability in the US. Although most stroke patients make some degree of recovery in weeks to months after the insult, it has become increasingly apparent that promoting recovery in the stroke patients requires appropriate rehabilitation therapy. Despite the progress made in animal research, clinical trials have generally failed to demonstrate the robust and significant improvement in patient outcome necessary for clinical application. Due to the complexity of our brain, multiple cascades of pathways are involved in secondary injury and recovery. There is a growing recognition that therapies targeting more than one pathway are needed to improve patient outcome. Our preliminary study suggests that netrin-1 gene therapy improves the neural structure and function. This proposed study will establish proof-of-concept evidences for future development of adjuvant therapeutics targeting netrin-1 in stroke rehabilitation.